Corneal opacity is a source of blindness for millions worldwide. Penetrating keratoplasty (PK) using cadaveric corneas is highly successful, but donated tissue is in limited supply and over time PK grafts are subject to a significant rate of complications and failure. Developing better therapy for corneal opacity is world health-care need that has inspired this project. In the 10 years of this project we have identified stem cells from human corneal stroma (CSSC) and showed that these cells (a) differentiate to keratocytes, (b) produce corneal stromal tissue in vitro, and (c) prevent formation of corneal scars in mouse models in vivo. The long-term goal of the project is develop stem cell-based therapeutic applications that can treat or reverse corneal scarring reducing the need for PK. In the renewal of the project we will work toward this goal with three specific aims. CSSC but no other stromal cells express N-cadherin (NCad). Aim 1 will test the hypothesis that NCad identifies a population of cells with keratocyte differentiation potential and provides a cell surface marker useful for isolating cells with high potential to effect corneal regeneration. We have found that CSSC can secrete connective tissue with aligned collagen and proteoglycans similar to corneal stroma. Aim 2 will test the role of microstructure topology and stiffness of solid substrates in directing the composition and organization of this CSSC-secreted matrix. The aim will also test the role of collagen crosslinking enzymes LOXL2 and TGM2 in increasing the strength and stiffness of the matrix elaborated by CSSC. This aim will assemble multilamellar constructs by stacking of the engineered tissue sheets. These experiments will provide insights on how the 3D environment directs secretion and organization of transparent stromal tissue. We have recently shown that CSSC instilled in healing corneal wounds prevent scarring and reconstitute ablated tissue with matrix indistinguishable from native cornea. Aim 3 will investigate how CSSC induce this tissue regeneration. CSSC reduce infiltration of neutrophils and induce the presence of TGF3, a cytokine attributed to induce scarless wound healing. This aim will test the hypothesis that via secretion of TSG-6 protein, CSSC reduce neutrophil infiltration and influence macrophages to adopt an alternative activated (M2) phenotype stimulating tissue regeneration by secretion of TGF3. The impact of these studies will be identification of novel molecular mechanisms controlling corneal stromal tissue matrix synthesis and organization. Of particular potential significance is identification of a mechanism to induce regeneration of native mammalian tissue. There will also be progress toward the goal of clinical use of CSSC. Thicker, stronger, and more organized tissue constructs will be produced with increased potential for use in lamellar grafts. Identification of properties linked to immunosuppressive and regenerative potential for CSSC will improve potential for use of these cells in cell-based therapy.